The present invention relates to the use of chemiluminescence in analytical determinations, for example, for immunossay.
Chemiluminescence is a physical phenomenon which is based on a chemical reaction and is therefore fundamentally different from fluorescence. In fluorescence, a molecule is excited by visible light or UV light, but remains unchanged and merely reradiates (emits) the absorbed light. The emission is either at the same wavelength as the absorbed light or a longer wavelength.
In contrast, the mechanism of chemiluminescence is based on the formation of high-energy, unstable intermediate products which decompose, with emission of light, to give an end product.
Analytical processes which utilize fluorescence are known per se, but background fluorescence has been found to affect seriously the results obtained, and has made it necessary to employ some expedient to eliminate this interfering fluorescence from the measurement.
Thus, according to German Offenlegungsschrift No. 2,628,158, the effect of background fluorescence is avoided by using as the labelling material only those fluorescent substances which, with appropriate excitation and at room temperature, have a narrow fluorescence spectrum coupled with a long decomposition time, and also by ensuring, by an appropriate scanning mechanism, that the measurement takes place only at a point in time when the interfering or competing fluorescence has already decayed, i.e. after about 100 nano-seconds.
The method disclosed in German Offenlegungsschrift No. 2,628,158 has, however, the disadvantages that the actual measurement, in each case, can only take place after the decay phase, and that relatively expensive equipment is required to couple the radiation pulse to the detection device so that the measurement is made not too early or too late.
A summary of the difficulties which occur in fluorescence immunoassay processes may be found in "Clinical Chemistry" (1979), pages 353-361.
Proposals to utilize chemiluminescence for quantitative analytical processes have been based on the property of, in particular, the organic compound known by the name luminol, i.e. that luminol reacts with oxygen in alkaline solution and thereby forms an unstable intermediate product which then decomposes, with emission of light. See, for example, U.S. Pat. No. 4,104,029, to Charles L. Maier, Jr., issued Aug. 1, 1978.
However, this method has not found acceptance in analytical practice, especially in immunoassay, probably mainly because when luminol is subjected to the chemical modification necessary to enable it to be coupled to proteins of the antigen and antibody type, its molecular structure is altered such that the reaction required for chemiluminescence is impaired. This view is also supported by the observations made by Simpson and co-workers (see, for example "Nature", Volume 279 (1979), pages 646/647) that the radiation intensity of diazotized luminol is only barely 1% of that of unmodified luminol.
A system in which fluorescein can be excited to luminescence by chemical processes is also described, in "Journal of Physical Chemistry", 78 (1974), pages 1681-1683.
In this reference, a system consisting of H.sub.2 O.sub.2 and NaOCl is used as a reactant for the chemiluminescence reaction. However, it was reported that the concentration of the fluorescein required for detection is extremely high. The smallest amount detected was 2.times.10.sup.-4 mol, so that, compared with the requirements of immunoassay, the sensitivity is at least six orders of magnitude too low. It was also reported that the life of the luminescence in the system mentioned is only 2.mu. s, that is to say, the emission of photons takes place instantaneously. These very rapid kinetics are completely unsuitable for sensitive measurement. Photon counters are used for detecting the photons because they are the most sensitive measuring devices, but because of their typical dead time of 20 ns, only limited maximum pulse rates can be processed. Quantitative and sensitive measurement of luminescence phenomena which takes place instantaneously is thus impossible.
In order to enable the chemiluminescence method to be applied to immunoassay, it would be desirable to achieve the following:
1. The sensitivity should be increased so that as little as 10.sup.-10 mol of the labelling agent can be detected.
2. The reaction kinetics should be changed so that the emission of light does not take place instantaneously but, for example, over a period of several seconds.
It would furthermore be desirable:
3. to initiate the luminescence not by the addition of two reagents (H.sub.2 O.sub.2 +NaOCl), but only by a single reagent. This would on the one hand simplify the measuring equipment (only one dispenser would be required, instead of two). Also, however, when two reagents are mixed, even in the absence of the chemiluminescent substance to be detected, considerable background luminescence is produced. This background is pronounced in the case of the system H.sub.2 O.sub.2 /NaOCl, and furthermore is not constant, so that there are again the disadvantages known in the case of fluorescence.